Osamu Ohno

Inhibition of Lipopolysaccharide Activity by a Bacterial Cyclic Lipopeptide Surfactin

Compounds that inactivate lipopolysaccharide (LPS) activity have the potential of being new anti-inflammatory agents. Therefore, we searched among microbial secondary metabolites for compounds that inhibited LPS-stimulated adhesion between human umbilical vein endothelial cells (HUVEC) and HL-60 cells. By this screening, we found a cyclic lipopeptide surfactin from the culture broth of Bacillus sp. BML752-121F2 to be inhibitory. The addition of the surfactin prior to the LPS stimulation decreased HL-60 cell-HUVEC adhesion without showing any cytotoxicity. We confirmed that surfactin inhibited LPS-induced expression of ICAM-1 and VCAM-1 in HUVEC. It also inhibited the cellular adhesion induced by lipid A, the active component of LPS; but it did not inhibit TNF-α or IL-1β-induced cell adhesion. Then, surfactin was shown to suppress the interaction of lipid A with LPS-binding protein (LBP) that mediates the transport of LPS to its receptors. Finally, surface plasmon resonance (SPR) analysis revealed the surfactin to interact reversibly with lipid A. Thus, this Bacillus surfactin was shown to be an inhibitor of LPS-induced signal transduction, directly interacting with LPS.

Bioactive secondary metabolites from symbiotic marine dinoflagellates: symbiodinolide and durinskiols.

Symbiotic relationships play critical roles in marine ecosystems. Among symbionts, marine dinoflagellates have attracted the attention of natural products chemists, biologists, and ecologists, since they are rich sources of unique bioactive secondary metabolites. The polyol compound symbiodinolide, which was isolated from the symbiotic dinoflagellate Symbiodinium sp., exhibits significant voltage-dependent N-type Ca(2+) channel-opening activity and may serve as a defense substance to prevent digestion of the host animals. Durinskiols are also unique long carbon-chain polyol compounds that were isolated from the dinoflagellate Durinskia sp. We found a selective cleavage reaction of allylic 1,2-diol using an olefin metathesis catalyst, and developed a fluorescent-labeling method for MS/MS analysis to achieve the structural elucidation of huge polyol compounds. This review highlights recent advances in structural and biological studies on symbiodinolide, durinskiols, and related polyol compounds.

Symbiopolyol, a VCAM-1 inhibitor from a symbiotic dinoflagellate of the jellyfish mastigias papua

A super-carbon-chain compound, symbiopolyol (1a), was isolated from a symbiotic dinoflagellate of the jellyfish Mastigias papua. Although a direct comparison between symbiopolyol (1a) and lingshuiol B has not been completed, symbiopolyol (1a) is suggested to be the enantiomer of lingshuiol B. The structure of 1a, including its partial relative configuration, was elucidated on the basis of interpretation of spectroscopic data and chemical transformations. This compound exhibited significant inhibitory activity against the expression of VCAM-1 in human umbilical vein endothelial cells (HUVEC).

Acetylcholine and norepinephrine concentrations in the heart of spontaneously hypertensive rats: a parasympathetic role in hypertension.

To determine the role of the parasympathetic system in the development of essential hypertension, the concentrations of acetylcholine (ACh) as a parasympathetic marker and norepinephrine (NE) as a sympathetic marker were measured simultaneously in the heart of spontaneously hypertensive rats (SHR) at 4, 10 and 27 weeks of age, and in age-matched control Wistar-Kyoto rats (WKY). ACh was measured using a highly sensitive and specific high performance liquid chromatography-electrochemical detection (HPLC-ECD) attached to a newly developed immobilized fixed enzyme column system. NE was measured by conventional HPLC-ECD. The ACh concentration decreased in the order right atrium (RA) greater than left atrium (LA) greater than right ventricle (RV) greater than interventricular septum (IVS) greater than left ventricle (LV) in both SHR and WKY of all age groups, and NE showed an almost identical pattern. The concentration of ACh tended to increase and that of NE to decrease with age in both strains. The concentrations of ACh in the heart were significantly different in the two strains, with the levels being consistently higher in the SHR strain regardless of age, although differences were most significant at 10 weeks. The mean concentrations of NE were higher in SHR than in WKY, but the differences between the two strains were significant only in the RV and IVS. The functional meanings of a given tissue concentration of a neurotransmitter are still controversial. However, the increase in SHR heart ACh concentration, in parallel with the development of hypertension, may reflect the augmented parasympathetic activity counteracting or compensating for the augmented sympathetic drive in the early stage of hypertension.

Inhibitory effects of bakuchiol, bavachin, and isobavachalcone isolated from Piper longum on melanin production in B16 mouse melanoma cells.

An EtOH extract of fruits of Piper longum was found to exhibit a potent inhibitory effect against α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 mouse melanoma cells. Bioassay-directed fractionation led to the isolation of prenylated phenolic compounds bakuchiol, bavachin, and isobavachalcone. These compounds and the crude extract of the fruits of P. longum may have suppressive effects against pigmentation by melanin in the skin.

Fluorescent Aplyronine A: Intracellular Accumulation and Disassembly of Actin Cytoskeleton in Tumor Cells

Actin is one of the abundant proteins in the cytoskeleton and is essential for the regulation of various functions, such as muscle contraction, cell motility, and cell division. Various actin-depolymerizing agents have been found in marine invertebrates, and some show extremely strong cytotoxicity. Among them, aplyronine A (ApA, 1, Figure 1), which was isolated from the sea hare Aplysia kurodai, has been shown to exhibit remarkable antitumor activities in vivo against P388 murine leukemia cells (T/C 545 %, 0.08 mg kg ) and several cancers. It depolymerizes fibrous actin (F-actin) and inhibits the polymerization of actin by forming a 1:1 complex with the monomeric globular molecule (G-actin, Kd 100 nm). [3] Studies on structure–activity relationships, X-ray analysis of the actin– aplyronine A complex, and photoaffinity labeling experiments have established the specific interactions of 1 with actin. However, the modes of action of ApA and related actintargeting natural products in tumor cells have not been well investigated, despite their great potential as preclinical candidates for use in cancer chemotherapy. We have recently developed a biotin derivative of 1 that exhibits potent cytotoxicity and causes actin disassembly in tumor cells, and have identified actin-related proteins 2/3 (Arp2 and Arp3) as presumed targets of 1. It was suggested that ApA might inhibit the ability of the Arp2/3 complex to bind to and branch F-actin. Also, it was shown that ApA (1) caused prominent caspase-dependent apoptosis in human leukemia HL-60 cells and human epithelial carcinoma HeLa S3 cells at sub-nanomolar concentrations. To explain the potent antitumor and apoptogenic effects of 1, we prepared its fluorescent derivatives and observed its dynamic behavior in living cell systems. Using these derivatives as molecular probes, we show here that ApA (1) caused the rapid disassembly of actin cytoskeleton, the malfunction of cell adhesions, and the dephosphorylation of focal adhesion kinase in tumor cells with apoptosis. Based on the finding that the C34 N-formyl enamide moiety of 1 can be replaced with hydrogen bond acceptors without a significant loss of activity, natural ApA (1) was hydrolyzed to give the C34 aldehyde, which was condensed with an oxyamine to afford tetramethylrhodamine-conjugated (TAMRAconjugated) ApA (ApA-FL, 2, Figure 1 and Figures S1 and S2 in the Supporting Information). Similarly, a TAMRA-conjugated derivative of aplyronine C (ApC-FL, 4) was prepared from ApC (3), an extremely minor congener of 1 in A. kurodai that lacks the C7 trimethylserine moiety, as well as a TAMRA-conjugated form of mycalolide B (MyB-FL, 6) from mycalolide B (5), an actin-depolymerizing tris-oxazole macrolide isolated from a Japanese marine sponge (Mycale sp.). For comparison, model TAMRA analogue 7 was also synthesized from 3-phenylpropionaldehyde. ApA-FL (2) showed a potent cytotoxicity against HeLa S3 cells (IC50 370 pm), whereas fluorescent derivatives 4 and 6 exhibited activities ca. 30 and 840 times weaker than that of 2. In an in vitro actin-depolymerizing assay, ApA-FL (2) significantly reduced the fluorescence of pyrene-labeled (pyrenyl) Factin (EC50 0.8 mm against 3 mm actin), and was more effective than 1 (EC50 1.3 mm), whereas model compound 7 scarcely exhibited this activity (Figure 2). Also, inhibition of F-actin sedimentation caused by 2 was directly detected by an SDS-PAGE analysis. On treatment with 1 or 2, the protein bands were observed almost entirely in the supernatant, as with G-actin (far left), which established that they had potent actin-depolymerizing properties. In contrast, 7 did not increase the amount in the supernatant, as with the control (second from left), which suggests that it does not depolymerize F-actin. Fluorescence microscopy observations revealed that the TAMRA derivatives 2, 4, 6, and 7 were all readily (less than 15 min) incorporated into HeLa S3 cells. Notably, ApA-FL (2) and ApC-FL (4) were retained well and distributed through the cytoplasm even after cells were washed with culture medium and incubated for an additional hour (Figure 1). We observed that ApC-FL (4) also accumulated through the nucleus, but that ApA-FL (2) did not at all. In contrast, model 7 was almost completely excluded from the cells under the same treatments. These significant differences in intracellular accumulation were established more clearly by flow cytometry analyses. The initial cellular incorporations of MyB-FL (6) and model 7 were less than that of 2 by factors of ca. 4–5. Furthermore, ApA-FL (2) was recovered as a unique TAMRA-containing component in HeLa S3 cells that had been treated for 1.5 h (Figure S3), which suggested that it exhibits strong bioavailability as well as highlighting the stability of the oxime bond. To visualize actin depolymerization in tumor cells, ApA-FL (2, 3 mm) was added to HEp-2 cells that expressed a green fluores[a] Prof. Dr. M. Kita, K. Yoneda, Y. Hirayama, K. Yamagishi, Y. Saito, Prof. Dr. H. Kigoshi Graduate School of Pure and Applied Sciences, University of Tsukuba 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571 (Japan) E-mail : mkita@chem.tsukuba.ac.jp kigoshi@chem.tsukuba.ac.jp [b] Y. Sugiyama, Dr. Y. Miwa Graduate School of Comprehensive Human Sciences, University of Tsukuba 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575 (Japan) [c] Dr. O. Ohno, M. Morita, Prof. Dr. K. Suenaga Faculty of Science and Technology, Keio University 3-14-1, Hiyoshi, Yokohama 223-8522 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200385.

Complanine, an inflammation-inducing substance isolated from the marine fireworm Eurythoe complanata

The marine fireworm, Eurythoe complanata, is known as a dangerous animal for humans because it induces skin inflammation through its small setae. Here, the inflammation-inducing substance was successfully isolated from the whole body using a methanolic extraction, and the inflammatory activity was determined using a bioassay. The structure was spectroscopically revealed to be a trimethylammonium with an unsaturated carbon chain and was named complanine. Complanine enhanced PKC activity in combination with TPA in vitro. This may explain the molecular mechanism behind its inflammation-inducing activity.

Apoptosis-inducing activity of the actin-depolymerizing agent aplyronine A and its side-chain derivatives.

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S(3) cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3-6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3-6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.

Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.

The duck-billed platypus (Ornithorhynchus anatinus) is one of the few venomous Australian mammals. We previously found that its crude venom potently induces Ca(2+) influx in human neuroblastoma IMR-32 cells. Guided by this bioassay, we identified 11 novel peptides, including the heptapeptide H-His-Asp-His-Pro-Asn-Pro-Arg-OH (1). Compounds 1-4 and 5-11 coincided with the 6-9 N-terminal residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP) and the 132-150 part of OvCNP precursor peptide, respectively. Heptapeptide 1, which is one of the primary components of the venom fluid (approximately 200 ng/microL), induced a significant increase in [Ca(2+)](i) in IMR-32 cells at 75 microM. To the best of our knowledge, this is the first example of the isolation of the N-terminal linear fragments of CNPs in any mammal.

Jahanyne, an apoptosis-inducing lipopeptide from the marine cyanobacterium Lyngbya sp.

An acetylene-containing lipopeptide, jahanyne, was isolated from the marine cyanobacterium Lyngbya sp. Its gross structure was established by spectroscopic analyses, and the absolute configuration was clarified based on a combination of chiral HPLC analyses, spectroscopic analyses, and derivatization reactions. Jahanyne significantly inhibited the growth of human cancer cells and induced apoptosis in HeLa cells.